Development of Antioxidant Packaging Material

by Applying Corn-Zein to LLDPE Film

in Combination with Phenolic Compounds
Hye-Yeon Park, Sung-Jin Kim, Ki Myong Kim, Young-Sun You, So Yeon Kim, and Jaejoon Han

Abstract: Functional active packaging materials were successfully developed by incorporating antioxidant agents into
corn-zein-laminated linear low-density polyethylene (LLDPE) film. The minimum effective concentrations of the active
compounds (for example, thymol, carvacrol, eugenol) were determined and these compounds were then laminated into
LLDPE films to develop corn-zein-laminated films with antioxidant agents. The release rate of antioxidant agents in

E: Food Engineering &

gas and liquid media were determined along with the mechanical and water barrier properties of the films containing

Physical Properties
these compounds. Tensile strength and percentage elongation at break were reduced in the corn-zein-laminated LLDPE
films when compared to typical LLDPE film. Furthermore, the ability of the corn-zein-laminated films to repel moisture
decreased by approximately 12.2%, but was improved by incorporating hydrophobic antioxidant compounds in the corn-
zein layer. Examination of release kinetics in the gas and liquid phases verified that antioxidants were effectively released
from the films and inhibited oxidation during testing. Finally, the films were used for fresh ground beef packaging,
and effectively inhibited lipid oxidation and had a positive effect on the color stability of beef patties during storage.
These results indicate that the developed antioxidant films are a novel active packaging material that can be effectively
implemented by the food industry to improve the quality and safety of foods.
Keywords: antioxidant film, corn-zein, diffusion test, ground beef, mechanical property

Practical Application: Zein protein, a by-product of corn processing industry, was laminated into plastic films in combi-
nation with natural phenolic compounds to develop antioxidant packaging films. The films demonstrated their efficient
release patterns of antioxidant compounds, which are suitable for packaging applications and food protection.

Introduction
The main purpose of food packaging is to maintain the qual-
ity and safety of food during distribution and storage. In recent
years, there have been increasing concerns about food quality and
safety, which have motivated the development of active packaging
(Dallyn and Shorten 1988). Active packaging is an innovative type
of packaging that prevents or slows deterioration in food quality
(Buonocore and others 2003). Oxygen scavengers, carbon diox-
ide scavengers/emitters, moisture absorbers and ethanol generators
are some examples of active packaging. Active packaging also in-
volves the release of antioxidants and/or antimicrobial substances
onto the food surface. Due to environmental and health concerns,
active packaging has been made from eco-friendly and biodegrad-
able food packaging materials combined with natural preservatives
(Appendini and Hotchikiss 2002; Suppakul and others 2003).
Polyethylene (PE), the most common and least expensive syn-
thetic polymer, is a widely used plastic material in food packaging.
MS 20120474 Submitted 3/28/2012, Accepted 7/10/2012. Authors Park,
S.-J. Kim, and Han are with Dept. of Food Science and Biotechnology, Sungkyunkwan
Univ., Suwon 440-746, Republic of Korea. Author K. M. Kim is with Dept. of Food
and Nutrition, Honam Univ., Gwangju 506-714, Republic of Korea. Author You
is with Bio Polymer R&D Center, Korea Bio Material Packaging Assn., Bucheon
421-742, Republic of Korea. Author S. Y. Kim is with Boimedical Research Institute,
Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea. Direct
inquiries to author Han (E-mail: han2009@skku.edu).
PE is the family name for resins such as low-density polyethy-
lene (LDPE), high-density polyethylene (HDPE), and linear low-
density polyethylene (LLDPE) (Robertson 1993). However, de-
spite the physical and economic advantages of PE films, they cause
environmental problems due to their nondegradability and diffi-
culty in recycling. Therefore, researchers have begun to use nat-
ural polymers as substitutions for synthetic polymers to develop
biodegradable food packaging materials (Peppas 2004).
Biopolymers are biodegradable, nontoxic, recyclable polymers
that are frequently applied as edible coatings or films (Cuq and
others 1998). They are made from natural sources such as polysac-
charides, proteins, and lipids. Zein is the major storage protein of
corn (Zea mays L.) and comprises 45% to 50% of corn protein.
It is a widely used biopolymer for producing biodegradable pack-
aging materials. The advantage of corn-zein is its ability to form
tough, relatively hydrophobic, and grease-proof films with excel-
lent flexibility and compressibility (Lai and others 1997). However,
corn-zein film has problems relating to its brittleness under dry
conditions, which restricts its use as a free-standing film or a coat-
ing material (Pomes 1971; Shukla and Cheryan 2001). To over-
come this problem, biopolymers are often coated on conventional
synthetic plastic films to develop a multilayered film structure (Atik
and others 2008).
For over 50 y, antioxidants have been added to the food-making
process to delay auto-oxidation (Cuvelier and others 1994). In ac-
tive antioxidant packaging, this consists of introducing antioxidants


C 2012 Institute of Food Technologists
R

doi: 10.1111/j.1750-3841.2012.02906.x Vol. 77, Nr. 10, 2012 r Journal of Food Science E273
Further reproduction without permission is prohibited
 Antioxidant packaging film . . .
into food packaging material instead of adding them directly to
the food (Tovar and others 2005). A wide range of compounds,
most of them synthetic molecules, have been used as antioxidant
agents in packaging (Padgett and others 1998). Currently, the most
frequently and widely used synthetic antioxidants in active food
packaging are butylated hydroxyanisole (BHA) and butylated hy-
droxytoluene (BHT), which are highly stable and efficient, and are
also cost-effective. Although these antioxidants can effectively in-
hibit the lipid oxidation of foods, the use of synthetic antioxidants
is strictly regulated as they are known to cause health risks (Branen
1975) and is becoming increasingly unacceptable to consumers be-
cause of their potential toxicity (Chan and others 2007; Yen and
others 2008). Due to safety concerns in the active food packaging
sector, natural compounds have been deemed more appropriate.
Essential oils, extracted from herbs or spices, are regarded as
E: Food Engineering &
natural alternatives to synthetic preservatives and the use of these
Physical Properties
oils in food meets consumer demands for safe food products (Burt
2004). Many essential oils exhibit antioxidant activity, which is
attributable to their high phenolic compound content. Phenolic
compounds are free radical scavengers, metal chelators, reducing
agents, hydrogen donors, and singlet oxygen quenchers (Proestos
and others 2006). In particular, thymol, carvacrol, and eugenol are
some of the most active phenolic antioxidants found in essential
oils. They are the major phenolic compounds present in thyme,
oregano (thymol and carvacrol), and clove (eugenol) essential oils.
Roberto and Baratta (2000) reported that thymol, eugenol, and
carvacrol exhibited the highest antioxidant activity in a study of
100 pure components of essential oils. In recent reports, eugenol,
menthol, and thymol maintained the quality and safety of sweet
cherries under modified atmospheric conditions (Serrano and
others 2005).
Active packaging acts as an inert barrier between food products
and the external environment, and the incorporation of antiox-
idant agents into polymeric packaging materials is an exciting
development in food packaging (Nerı́n and others 2006). Antiox-
idants are released from packaging films by diffusion, and directly
inhibit lipid rancidity on the food surface, especially in oxidation-
sensitive foods. Antioxidant packaging also has the potential to
extend the shelf life of foods (Anklam and others 1997; Morales-
Aizpuruaand and Tenuta-Filho 2005).
The objective of the present study was to develop multilayer
films based on corn-zein and LLDPE films incorporated with an-
tioxidant agents (thymol, carvacrol, and eugenol). The mechanical
properties and hydrophobic qualities of developed films and the
release rate of antioxidant agents from the film matrix into model
systems were measured. In addition, the effectiveness of the de-
veloped packaging films on color stability and lipid oxidation for
the storage of fresh ground beef was evaluated.
Materials and Methods
Chemicals and reagents
Corn-zein, antioxidant agents (thymol, carvacrol, and eugenol),
and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from
Sigma-Aldrich Chemicals Co. (St. Louis, Mo., U.S.A.). Polyethy-
lene glycol 400 (PEG 400), a plasticizer of corn-zein film, was
obtained from Junsei Chemicals Co. Ltd. (Tokyo, Japan). LLDPE
film was obtained from Hanjin P&C Co. Ltd. (Seoul, Korea), while
propyl gallate (PG) and ethylenediaminetetraacetic acid (EDTA)
were obtained from DAEJUNG (Siheung, Korea). Thiobarbituric
acid (TBA) was purchased from Alfa Aesar (St. Ward Hill, Mass.,
U.S.A.). Figure 1–Structure of zein-laminated LLDPE film with antioxidant agent.
E274 Journal of Food Science r Vol. 77, Nr. 10, 2012
Film preparation
Corn-zein coating solutions were prepared according to the
methods described by Mastromatteo and others (2009) with some
modifications. Corn-zein was dissolved in 95% ethanol at 20 g/
100 mL. As a plasticizer, PEG 400 was added to corn-zein solu-
tion at 4 g/100 mL and the solution was then heated at 70 ◦ C for
15 min. After cooling, thymol, carvacrol, and eugenol were in-
dividually added to the corn-zein solutions at a designated con-
centration (1.5, 3, or 5%, v/v) and stirred for 15 min. Corn-zein
solutions containing different concentrations of each antioxidant
agent were coated onto corona-treated LLDPE films (surface ten-
sion 45 ± 5 dyne/min; thickness 40 ± 0.4 μm) using a Nr. 28
coating rod (RDS, Webster, N.Y., U.S.A.) and dried for 15 min
at room temperature. The coated side of the double-layered film
was laminated with another LLDPE film using a heat laminat-
ing machine (GMP Co., Paju, Korea). The final multilayered film
(thickness 95 ± 2.2 μm) structure is shown in Figure 1.
Measurement of film properties
Film thickness. Laminated film thickness was measured with
a digital micrometer (ID-C112X, Mitutoyo, Kawasaki, Japan) at
5 random positions on the film. The mean of the 5 measure-
ments was used to calculate tensile strength (TS) and water vapor
permeability (WVP).
Mechanical properties. Mechanical properties of the films
were determined by ASTM standard method D882-91 (1995)
using an Instron universal testing machine (Model 5566, Instron
Engineering Co., Canton, Mass., U.S.A.). Films were cut into
strips with a test dimension of 25.4 mm × 100 mm. Before testing,
all of the films were conditioned for 48 h in a thermo-hygrostat
(LAM-MADE011, Sejong Scientific Co., Bucheon, Korea) held
at 25 ◦ C and 50% RH. The equilibrated strip was placed between
grip heads of the machine. The initial grip separation and cross
head speed were set at 50 mm and 500 mm/min, respectively. TS
was expressed in MPa and was calculated by dividing the maximum
load (N) by the initial cross sectional area (m2 ) of the specimen.
The percentage elongation at break (%E) was calculated as the
ratio of the final length at the point of break to the initial length
of a specimen (50 mm) and expressed as a percentage. TS and %E
were replicated 5 times for each type of film.
Water vapor permeability. WVP was determined gravimet-
rically, based on ASTM standard method E96-95 (1997). Each film
sample was mechanically sealed onto a polymethylmethacrylate
cup containing 30 g of anhydrous calcium chloride. The weights
of the sealed cups were measured using an electronic scale with a
precision of 0.001 g and cups were left in a controlled humidity
chamber adjusted to 30 ◦ C and 90% RH. After 24 h, they were
reweighed and WVP was calculated according to the following
Antioxidant packaging film . . .
formula:
 
WVP g · mn/m2 · d · kPa = W · x/A · T (P2 − P1 ) ,
where W is the increase in weight of the cup (g) after 1 d, x is
the average thickness of the film (mm), A is the exposed area of
the film (1.66 × 10−3 m2 ), and (P 2 − P 1 ) is the differential vapor
pressure across the films (P 2 − P 1 = 1.70 kPa at 30 ◦ C). WVP
measurements of each film sample were carried out in triplicate.
Release characteristics in different media
Release of antioxidant agent into the atmosphere. The
release test was carried out according to the methods described
in Gamege and others (2009), with some modifications. Corn-
zein-laminated LLDPE films with antioxidant agent were put into
a plastic tube (500 mL volume) and carefully adhered to the in-
ner wall to avoid gaps. The tube was tightly sealed with a cap
and stored at room temperature. Air samples (1 mL) were peri-
odically withdrawn through a rubber septum on the tube using
a syringe to determine the release of antioxidant agent from the
films into the inner atmosphere. The amount of antioxidant in the
gaseous medium was analyzed using the 6890A gas chromatog-
raphy (GC) system (Agilent Technologies, Santa Clara, Calif.,
U.S.A.) equipped with an FID detector and HP5 MS capillary
column (30 m × 0.25 mm× 0.25 μm). Gaseous samples (1 mL)
were injected manually, and the split ratio was 1:60. Helium was
used as the carrier gas at a flow rate of 1 mL/min. The split/splitless
injector temperature and detector temperature were set at 200 ◦ C
and 350 ◦ C, respectively. The initial column temperature was set
at 60 ◦ C and increased to 200 ◦ C at a rate of 10 ◦ C/min. The
antioxidant compound was identified by comparing its mass spec-
tra with those of authentic compounds in the computer library.
The concentration of the identified compound in the atmosphere
was quantified according to the peak area integrated by the data
analysis program. Tests were conducted 4 times each.
Release of antioxidant agent into the liquid state. The
release of antioxidant agent from the films into liquid medium
was measured by 2,2-DPPH radical scavenging assay according
to methods described in Shimada and others (1992), with some
modifications. The films were tightly adhered to the inner wall of
a 30 mL glass vial filled with 25 mL of 0.1 mM DPPH solution.
The vial was wrapped with aluminum foil thoroughly to avoid
light, and then stored in a shaking incubator (Vision Scientific Co.
Ltd., Korea) at 25 ◦ C and 70 rpm. The absorbance was measured
with an Ultrospec 3100 Pro UV spectrophotometer (Pharmacia
Biotech Inc., Little Chalfont, U.K.) at 517 nm every 3 h. The test
directly measured the antioxidant activities of the released agent
into solution. DPPH radical scavenging activity was expressed as
milligrams of ascorbic acid equivalents (AAE) per grams of film
sample. Tests were replicated 4 times.
Effects of antioxidant packaging on ground beef patties
Beef samples and storage conditions. Fresh ground beef
(top round roast) was obtained from a local butcher shop in
Suwon, Korea, and stored under refrigerated conditions (4 ±
1 ◦ C). On the day of purchase, the roasts were trimmed of all
separable fat and connective tissue, ground twice, and then ana-
lyzed for fat content according to the methods described in Folch
and others (1957). Beef patties (30 g, 3.8% fat) were prepared us-
ing Petri dishes (6 cm dia, 1.2 cm height) in the laboratory for
each experiment. The raw beef patties were then packaged in 1
of 3 different ways, as follows: (i) over-wrapping with oxygen-
permeable polyvinyl chloride (PVC) film (O2 transmission rate =
91.54 cm3 /μm/m2 /24 h at 25 ◦ C), (ii) vacuum packaging with
LLDPE film, or (iii) vacuum packaging with developed antioxi-
dant films (0.3% or 3% antioxidant concentration). Samples were
stored at 4 ◦ C and the degree of lipid oxidation and the colors of
the patties in each packaging group were measured every 2 d.
Evaluation of lipid oxidation. Lipid oxidation of beef patties
was determined by analysis of 2-thiobarbiutric acid reactive sub-
stances (TBARS) according to the modified distillation method
described in Rhee (1978). To prevent further oxidation, a mixed
solution of PG and EDTA was added to each beef patty before
distillation. After adding 4N HCl solution and distilled water, the
mixture was distilled in a Kjeldahl flask and the distillate was col-
lected in a 50 mL Falcon tube. Then, the distillate was reacted
with 2-TBA in a water bath at 100 ◦ C for 35 min to induce color
E: Food Engineering &
Physical Properties
change. The optical density of the sample was read at 530 nm
with an Ultrospec 3100 pro UV spectrophotometer (Pharmacia
Biotech Inc.). The results of TBARS content were expressed as
milligrams of malondialdehyde equivalents (MDA) per kilogram
of meat sample. Tests were replicated 4 times.
Color changes in beef patty. Color changes were measured
with a CR-400 colorimeter (Minolta Co., Japan). The patties
were exposed to oxygen for 10 min before color evaluation. The
colorimeter was calibrated using a standard white plate. Measure-
ments were taken 5 times for each sample, and the mean values
were used to determine the color coordinates L ∗ (lightness), a∗
(redness), and b∗ (yellowness). In order to monitor meat browning
during storage, hue angle (H = arctan b∗ / a∗ ; higher values are
more brown) was calculated. Tests were conducted 4 times.
Statistical analysis
Data analysis was performed using Statistical Analysis System
(SAS) software, version 8.1 (SAS Inst., Cary, N.C., USA). The
General Linear Models Procedure was used for analysis of variance,
with main effect means separated by Student-Newman-Keuls test.
Significance was defined as P ≤ 0.05.
Results and Discussion
Mechanical properties of film
The mechanical properties of the corn-zein laminated LLDPE
films were measured by uniaxial tensile tests. The TS and %E of
laminated films were determined from the attained stress–strain
curves (Table 1).
The TS of simple LLDPE film was 31.33 MPa, which was
the highest value among all tested films. When comparing to
the simple LLDPE film, the TS of the corn-zein-laminated film
(LLDPE/zein/LLDPE) decreased (P ≤ 0.05) by 28.6% (22.38
MPa). The lower TS of the LLDPE/zein/LLDPE film was prob-
ably due to the physical weakness of the zein film layer. Rhim and
others (1997) reported that the TS of simple zein film was 5.52
MPa, which is much lower than LLDPE film. In zein-laminated
film groups, laminated films containing antioxidants had TS values
ranging from 22.28 to 24.27 MPa. Thus, the addition of an an-
tioxidant agent up to 5% (v/v) in the zein layer resulted in a slight
but insignificant increase in TS (P > 0.05). Generally, the TS of
laminated films is dependent on the substrate or base film rather
than the coating or laminated layer (Hong and others 2004). In
this study, zein film was applied to LLDPE film as a coating mate-
rial; LLDPE film was the substrate or base film in these laminated
films. In this study, the TS of zein-laminated LLDPE film was
Vol. 77, Nr. 10, 2012 r Journal of Food Science E275
 Antioxidant packaging film . . .

22.38 MPa. Earlier studies reported that zein-coated polypropy-
lene film had a TS of 45 MPa (Lee and others 2008), and the TS
of zein-coated carrageenan films ranged from 26.38 to 37.73 MPa
according to the concentration of zein-coating solutions (Rhim
and others 1997). This difference may be attributed to the different
substrate materials of the films.
The value of %E is associated with the elasticity and flex-
ibility of the film. Values of %E for simple LLDPE and
LLDPE/zein/LLDPE films were estimated to be 776.60% and
667.30%, respectively. Values of %E for LLDPE/zein/LLDPE film
decreased by 14% in comparison to LLDPE film, but slightly in-
creased when antioxidant agents were added. Gamage and oth-
ers (2009) reported that the %E values of various essential oil-
incorporated films were significantly higher compared to those
of nonessential oil-incorporated films. These results indicate that
E: Food Engineering &
LLDPE film had a lower WVP (1.778 g·mm/m2 ·d·kPa) than any
other laminated film, indicating that LLDPE film provides a good
barrier for moisture. In contrast, LLDPE/zein/LLDPE film had
the highest WVP at 2.033 g·mm/m2 ·d·kPa, and thus performed
the most poorly as a moisture barrier among the film samples
tested in this study. Results showed that simple zein-laminated
films had relatively high WVP compared to LLDPE film, which
was probably due to the hydrophilic groups in zein molecules.
These hydrophilic groups are formed from the amino acids that
compose corn-zein protein, and that they weakened the water bar-
rier properties of corn-zein-laminated film. The WVP of other
laminated films containing phenolic antioxidant agents were es-
timated between 1.913 and 1.958 g·mm/m2 ·d·kPa. Regardless
of the type of antioxidant agent used at 3% concentration (v/v),
films containing antioxidants had improved water barrier prop-

antioxidant agents act as adhesives in the protein polymer matrix, erties when compared to simple zein-laminated film due to the
Physical Properties

similar to plasticizer. Therefore, the addition of essential oil im-
proved the flexibility and %E of the films. Based on these results,
antioxidants may be considered as potential replacements for plas-
ticizers in protein biopolymer films. In addition, antioxidants can
contribute positively to the mechanical properties of packaging
materials composed of biopolymer.
Water barrier properties of films
WVP is determined by the chemical arrangement of film-
forming polymers and the morphology of the film (Siripatrawan
and Harte 2010). WVP was calculated according to the aforemen-
tioned formula, and the results are depicted in Table 2. Simple
strong hydrophobicity of the added antioxidant agents, which in-
terrupts the penetration of water molecules. Thymol, carvacrol,
and eugenol, which were used as antioxidant agents in this study,
are hydrophobic and are major compounds of naturally occurring
essential oils in thyme, oregano, and cloves. According to Sánchez-
González and others (2011), the same behavior was observed when
essential oils (bergamot, lemon, or tea tree oil) were incorporated
into chitosan/hydropropylmethylcellulose composite films. They
reported that WVP values of composite films showed a decrease
in line with the increase in the concentration of hydrophobic an-
tioxidant agent. The results of this study were in accordance with
these observations.

Table 1– Mechanical properties of zein-laminated LLDPE films

with different antioxidant compounds and concentrations. Release into the atmosphere
GC analysis was used to measure changes in the concentration
Film Tensile Elongation
construction strength (MPa) at break (%) of antioxidants released from the developed films into the atmo-
sphere (Figure 2). Overall, the maximum concentration of antiox-
LLDPE 31.33 ± 0.98a 776.60 ± 36.19a
idants in the headspace was correlated with the concentrations of
LLDPE/zein/LLDPE 22.38 ± 1.80b 667.30 ± 50.88b
LLDPE/zein with thymol 22.78 ± 2.90b 767.54 ± 63.66a antioxidants added to corn-zein film solution, and the released
1.5%/LLDPE concentrations of antioxidants reached a maximum within 2 d in
LLDPE/zein with carvacrol 24.21 ± 2.29b 746.03 ± 54.78a all experimental groups. Films containing higher concentrations
1.5%/LLDPE of antioxidants also had faster diffusion rates of antioxidants into
LLDPE/zein with eugenol 23.88 ± 1.81b 786.13 ± 61.11a
1.5%/LLDPE the air yet still maintaining higher concentrations of antioxidants
LLDPE/zein with thymol 3%/LLDPE 23.02 ± 1.73b 802.20 ± 35.17a than films with lower concentrations.
LLDPE/zein with carvacrol 23.14 ± 2.05b 744.47 ± 85.85a GC analyses of headspace gas composition revealed that the
3%/LLDPE concentrations of diffused antioxidants in the container reached
LLDPE/zein with eugenol 23.58 ± 2.01b 763.87 ± 82.10a
saturation after approximately 1 to 2 d, after which the amount
3%/LLDPE
LLDPE/zein with thymol 5%/LLDPE 22.83 ± 1.09b 786.27 ± 74.44a of antioxidants was reduced or maintained. Groups containing
LLDPE/zein with carvacrol 24.27 ± 1.95b 784.73 ± 55.54a thymol and carvacrol displayed a similar quantitative trend. There
5%/LLDPE were no significant changes in the concentration of antioxidants
LLDPE/zein with eugenol 22.28 ± 2.54b 784.33 ± 68.44a in these groups after day 2, indicating that all antioxidants escaped
5%/LLDPE
from these films within 2 d.
a,b
Within a column, different superscripts indicate significant differences (P ≤ 0.05).
The diffused antioxidants of zein-5% eugenol-laminated film
displayed the highest concentration and the longest holding time
Table 2– Water vapor permeability of zein-laminated LLDPE in the gas state. In this film, concentration of eugenol remained at
films with different antioxidant compounds. a high level for approximately 3 d and decreased over the following
Film Water vapor permeability 2 d. From day 5 to day 7, eugenol levels inside the headspace re-
construction (g·mm/m2 ·d·kPa) mained constant. The maximum value of this group was observed
up to day 3, and then it gradually reduced over last time pe-
LLDPE 1.778 ± 0.045a
LLDPE/zein/LLDPE 2.033 ± 0.112b riod. This might suggest that eugenol leaked out of the container
LLDPE/zein with thymol 3%/LLDPE 1.958 ± 0.105ab through the lid or septum of the container, even though the con-
LLDPE/zein with carvacrol 3%/LLDPE 1.913 ± 0.045ab tainer was sealed carefully. However, LLDPE/zein/LLDPE films
LLDPE/zein with eugenol 3%/LLDPE 1.920 ± 0.112ab containing 1.5% and 3% eugenol showed a similar pattern, even
a,b
Within a column, different superscripts indicate significant differences (P ≤ 0.05). though they contained different initial amounts of antioxidants.

E276 Journal of Food Science r Vol. 77, Nr. 10, 2012

Antioxidant packaging film . . .
Release into the liquid state
DPPH radical assays were performed with some modifications,
to evaluate the effectiveness of the antioxidant agents released
from the films into the liquid medium. DPPH radical scavenging
activities of the samples consistently increased with storage time
in all of the experimental groups, suggesting that there was a
continuous release of antioxidant agents (Figure 3).
There were slight changes in the radical scavenging effects of
LLDPE film and zein-laminated film even though they did not
contain any antioxidant agent. These changes might have been
Figure 2–Concentration changes of thymol (A), carvacrol (B), and eugenol
(C) released by antioxidant films into the atmosphere over 1 wk.
caused by the instability of DPPH radicals resulting in a grad-
ual extinction of radicals over time. By contrast, zein-laminated
films containing antioxidant (thymol, carvacrol, or eugenol) dis-
played considerable changes in the activity of released antioxidants.
Antioxidant film with eugenol had the maximum DPPH radical
scavenging activity of all the tested films. In addition, the reac-
tion mechanism of DPPH radicals and eugenol molecules released
from the film was terminated much sooner than thymol and car-
vacrol, at a concentration one-tenth that of the others. After 21 h,
absorbance at 517 nm of eugenol-containing film dropped below
0.1, which is a value that was too small to be measured correctly
by a UV spectrophotometer with the optimum measurable ab-
sorbance range of 0.1 to 2.0. Therefore absorbance of this film
was not evaluated after 21 h. The DPPH radical scavenging activi-
ties of thymol and carvacrol were similar, although thymol activity
E: Food Engineering &
was not significantly higher than that of carvacrol, perhaps because
Physical Properties
they are isomers with the same molecular formula and a similar
chemical nature.
Generally, the release of an active compound from the swelled
polymeric network occurs in 2 steps. First, water molecules pene-
trate into the polymeric matrix, causing the matrix to swell. Then,
active compounds diffuse through the widened mesh of the poly-
meric network (Del Nobile and others 2008). Therefore, release
of an active compound is affected by the structural properties of
the polymer or polymeric network and the diffusion rate of the
active compound. However, in this study, the structural proper-
ties of the films were likely not factors because the antioxidant
active films had the same structure as the LLDPE/zein/LLDPE
film. Moreover, the 3 antioxidant compounds added to the de-
veloped films have similar molecular weights and water solubility,
and therefore differences in diffusion rates were also negligible.
Consequently, the factor that influenced DPPH radical scaveng-
ing activities the most was the antioxidant ability of the antioxidant
agent. Yanishilieva and others (1999) reported that thymol is a bet-
ter antioxidant than carvacrol due to the greater steric hindrance
of the phenolic group in thymol. According to Mastelić and others
(2008), eugenol exhibited outstanding DPPH radical scavenging
activity, followed by thymol and carvacrol. This study reported
similar results.
TBA values
In the diffusion test of antioxidants in a liquid state, zein-
laminated film with 0.3% eugenol showed the greatest antioxidant
Figure 3–Diffusion of antioxidant compounds from the film to liquid medium
over 30 h.
Vol. 77, Nr. 10, 2012 r Journal of Food Science E277
 Antioxidant packaging film . . .

properties. Therefore, this film formulation was used for raw beef
patty packaging in this study. TBA value was measured in or-
der to examine the preventive effects of antioxidant-containing
films on lipid oxidation of meat during storage (Figure 4). In
all experimental groups, TBARS contents increased consistently
throughout storage. The control group, over-wrapped with the
oxygen-permeable PVC film, had a higher TBA value (P ≤ 0.05)
than all of the vacuum-packaged groups. In contrast, vacuum
packaging effectively protected beef patties from the beginning of
storage, with a TBA value lower than 2 mg MDA/kg over the
course of 14 d. There were few differences in TBA values among
the vacuum-packaged groups, and vacuum-packaged patties with
zein-laminated film with 3.0% eugenol had the lowest TBA val-
ues. Even though vacuum packaging alone inhibited lipid oxida-
tion effectively, the developed antioxidant films demonstrated a
E: Food Engineering &
improved the efficacy of vacuum packaging and stabilized raw
beef patties against oxidation. The antioxidant property of pre-
pared film containing 3% eugenol was similar to that of the film
containing 0.3% eugenol, which suggests that eugenol is a strong
antioxidant and 0.3% concentration was sufficient to completely
retard lipid oxidation in beef patties.
The inhibitory effect of eugenol on lipid oxidation is induced
by the phenolic group, which contains an electron-repelling group
in the ortho-position. This phenolic group is capable of reducing
both lipid radicals and ferric ion (Dorman and others 2000). Ogata
and others (1997) suggested that eugenol prevents lipid oxidation
by trapping active oxygen species, such as O2 − or hydroxyl radicals.
Lipid oxidation has been associated with off-flavors and off-
taste in meat and meat products. According to Insausti and others
(2001), humans detect off-flavors or off-taste in meat when TBA

synergistic effect in combination with vacuum treatment against values are greater than or equal to 5 mg MDA/kg. In this study,
Physical Properties

lipid oxidation, and completely suppressed lipid oxidation during
the 14 d. Specifically, TBA value of “vacuum only packaging”
increased 86.1% after 14 d, but TBA values of “vacuum packaging
with 0.3% eugenol-containing film” and “vacuum packaging with
3% eugenol-containing film” increased 40.9% and 27.7%, respec-
tively, after 14 d. This result implied that the antioxidant films
Figure 4–Effects of packaging material on the TBARS values of beef patties packaged samples, regardless of eugenol existence within films.
after 14 d.
TBA values above 5 mg MDA/kg meat were only observed in
the PVC-wrapped control group. In the other vacuum-packaged
groups, TBA values remained below 2 mg MDA/kg at all times
during storage.
Color changes
Color values including L ∗ (lightness), a∗ (redness), and b∗
(yellowness) of the oxygen-permeable control group and the
vacuum-packaged groups using antioxidant active films were mea-
sured and used to calculate the hue index. The color of meat is
one of the most important factors in customer selection because
color is an indicator of freshness (Mancini and Hunt 2005). Color
changes during storage at 4 ◦ C are presented in Table 3.
Treatment and storage time had no significant effects on L ∗ and
∗
b values of beef patties. In the control group, beef patty samples
suffered considerable decreases in a∗ values, in contrast to the other
groups which exhibited relatively high a∗ values. Vacuum packag-
ing and the addition of eugenol made it possible to maintain initial
a∗ levels over time. Allen and Conforth (2010) also observed that
after 14 d, the a∗ value of beef patties mixed with 0.05% eugenol
was significantly more reddish, and therefore more desirable to
consumers, than the control group. Control samples had signif-
icantly higher (P ≤ 0.05) hue angle values (more reddish and
less yellow) in comparison with the hue angle values of vacuum-
A significant increase in the hue angle value of a control sample

Table 3–Color value of beef patties packaged with the developed film.
Time of storage (d)
Color value Type of packaging 0 4 7 14
L ∗
PVC wrap 36.44 ± 2.57Aa
36.01 ± 1.93 Aa
36.77 ± 2.22 Aa
36.88 ± 1.52Aa
vacuum packaging only 38.09 ± 3.10ABa 36.81 ± 3.31ABa 35.77 ± 2.34Aa 36.19 ± 2.44ABa
vacuum packaging with antioxidant film (0.3% eugenol) 36.77 ± 3.71ABa 34.59 ± 2.27Ba 36.00 ± 2.25Aa 34.75 ± 2.00Ba
vacuum packaging with antioxidant film (3% eugenol) 34.96 ± 1.87Ba 34.06 ± 1.87Ba 34.76 ± 1.37Aa 35.36 ± 1.60ABa
a∗ PVC wrap 13.57 ± 2.07Aa 9.60 ± 1.36Ab 10.05 ± 1.94Ab 9.35 ± 1.17Ab
vacuum packaging only 12.19 ± 0.97Ba 12.99 ± 1.06Bab 13.94 ± 1.10Bb 12.86 ± 1.94Bab
vacuum packaging with antioxidant film (0.3% eugenol) 12.01 ± 1.01Ba 13.80 ± 1.80Bb 13.64 ± 1.42Bb 12.94 ± 1.40Bab
vacuum packaging with antioxidant film (3% eugenol) 12.63 ± 1.19ABa 13.52 ± 0.69Ba 13.68 ± 1.23Ba 11.26 ± 2.08Cb
b∗ PVC wrap 4.49 ± 1.17Aa 4.62 ± 0.75Aa 3.43 ± 0.82Ab 3.32 ± 0.78Ab
vacuum packaging only 3.61 ± 1.50Ba 3.73 ± 1.22Ba 3.52 ± 0.57Aa 3.85 ± 1.68Aa
vacuum packaging with antioxidant film (0.3% eugenol) 2.80 ± 0.81Ba 3.25 ± 0.72Bab 3.12 ± 0.65Aab 3.81 ± 1.13Ab
vacuum packaging with antioxidant film (3% eugenol) 2.98 ± 0.60Ba 3.00 ± 0.67Ba 3.36 ± 0.90Aa 2.79 ± 1.01Aa
Hue angle PVC wrap 18.09 ± 2.05Aa 24.30 ± 2.85Ab 25.27 ± 3.14Abc 27.20 ± 2.78Ac
vacuum packaging only 14.54 ± 2.66Ba 15.23 ± 1.70Ba 14.58 ± 1.19Ba 16.19 ± 2.55Ba
vacuum packaging with antioxidant film (0.3% eugenol) 13.06 ± 1.66Ba 13.39 ± 1.57Ca 12.83 ± 1.67Ba 15.42 ± 2.03Bb
vacuum packaging with antioxidant film (3% eugenol) 13.24 ± 2.14Ba 13.52 ± 1.62Ca 13.40 ± 1.69Ba 14.33 ± 2.52Ba
AtoC
: Within a column, different letters indicate significant differences (P ≤ 0.05).
a,b
: Within a row, different letters indicate significant differences (P ≤ 0.05).

E278 Journal of Food Science r Vol. 77, Nr. 10, 2012

Antioxidant packaging film . . .
indicated discoloration. The control group became brownish (de-
creased redness and increased yellowness) with an increase in stor-
age time. Color change in the control group might be associated
with lipid oxidation and the formation of metmyoglobin (Djenane
and others 2002; Nerı́n and others 2006). Beef patties vacuum-
packaged with antioxidant films showed lower changes in hue
angle values. In these groups, the films acted as an oxygen gas bar-
rier preventing the direct contact of meat with oxygen. Moreover,
the strong antioxidant property of eugenol released from the films
contributed to protection of the beef patties from lipid oxidation.
These data suggest that diffusion of eugenol from the packaging
delays oxidation of beef color pigments.
Conclusion
We developed zein-laminated LLDPE films with natural an-
tioxidants and measured their physical and functional properties.
In addition, diffusion modes of antioxidants in the gas and liquid
states were estimated. Zein-laminated films with antioxidants had
reduced TS and %E values compared to LLDPE films. These films
also had higher WVP than LLDPE film. The antioxidant film was
tested in fresh beef patty packaging. Antioxidant films effectively
retarded lipid oxidation and inhibited color change in beef during
storage. These results suggest that zein-laminated films contain-
ing antioxidants are suitable for packaging applications and food
protection.
Acknowledgment
This work was supported by the Natl. Research Foundation
of Korea (NRF) grant funded by the Korea government (MEST)
(Nr. 2011-0014710).
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